PROJECT SUMMARY Parkinson?s Disease (PD) is the second most common neurodegenerative disease in the world with a prevalence estimated to be approximately 1% in people over age 60, making it an increasingly important medical problem in our aging population. Levodopa is the current standard of care for PD and functions by increasing levels of dopamine centrally in the dopamine-depleted nigrostriatal pathway. Levodopa, however, is not a viable agent for long-term therapeutic use as undesirable side effects, notably motor fluctuations termed Levodopa-induced dyskinesias (LIDs), are commonly reported. LIDs are estimated to occur in over 50% of PD patients after 5 to 10 years of treatment and disproportionately impact older PD patients at more advanced disease stages by substantially limiting the therapeutic options for this population subset. Recent research has elucidated that, in addition to G protein signaling, dopamine receptors can also signal through a distinct b-arrestin2 (b-arr2)-dependent pathway. This pathway is important in regulating downstream responses at the Dopamine 1 Receptor (D1R) and plays a significant role in converting dopamine signaling into motor function. Previous studies showed that genetic modulation of b-arr2 signaling at D1R in rodent and non-human primate PD models improved motor functioning, while preventing LIDs. The differential activation of these distinct downstream signaling pathways by a ligand is termed ?functional selectivity? or ?biased agonism?, and to date, the ?-arr2 signaling pathway at D1R has not been pharmacologically targeted in a functionally selective manner to potentially reduce the adverse effects associated with Levodopa. To accomplish this, Aim 1 proposes the synthesis and characterization of new D1R ligands by systematically modifying a previously identified D1R-selective, non-catechol lead compound. Ligands will be profiled for functional selectivity using three assays that detect their relative potencies at activating G protein and b-arr2 signaling at D1R. The blood-brain barrier permeability of each ligand will be assessed using a validated artificial membrane assay. In Aim 2, in silico docking studies, molecular dynamics-based free- energy calculations, and model validation experiments utilizing previously synthesized analogues with diverse functional selectivity profiles will be performed to identify D1R structural elements that are important for ?-arr2 bias. These results will provide critical structural information to help deduce a structural mechanism for ?-arr2 recruitment at D1R and a structure-functional selectivity relationship (SFSR) that will inform further scaffold optimization into a potent, ?-arr2-biased compound. Findings from this project will greatly advance knowledge in the field by providing chemical tools that will enable the study of how biased signaling occurs at D1R and how molecular pathways downstream of this receptor contribute to PD and LID pathophysiologies. The insights gained from such studies will provide important clues guiding novel therapeutic approaches to this challenging, unsolved medical problem. !